PROJECT SUMMARY/ABSTRACT The mosquito-borne Zika virus (ZIKV), which the WHO has declared a global emergency, is spreading explosively in the Americas and as many as 4 million people could become infected by the end of 2016. From a global health perspective, the severe microcephaly and associated birth defects arising from infection make development of effective therapeutic agents for ZIKV paramount. The goal of this proposal, written in response to the recent call titled Rapid Assessment of Zika Virus (ZIKV) Complications (R21) (PAR-16-106) is to identify small molecule drug-leads that inhibit ZIKV entry and replication. Our overall objective is to develop inhibitors capable of blocking key conformational changes required for membrane fusion that involve the ZIKV envelope glycoprotein (E). We hypothesize that structure-based computational screening using knowledge of viral interfaces followed by experimental characterization will be more successful than random screening. The rationale for proposing the work, is the ability of our team to rapidly leverage prior experience gained targeting analogous events in HIV and Ebola and the ability to construct robust structural models for ZIKV screening based on the high homology of dengue virus glycoprotein E (74% similarity, 54% identify) for which abundant crystallographic structural information is available. The project is arranged as two Specific Aims according to the expertise of each PI's lab: (Aim #1) Identify and exploit targetable events in ZIKV glycoprotein E. Under this aim we will computationally target favorable protein interfaces to disrupt membrane fusion using high- throughput-virtual screening of ca. 5 million commercially available compounds. Three distinct E protein sites will be targeted, based on ZIKV models derived from related protein structures of dengue virus reported by Harrison and coworkers, include the previously described ?-OG pocket (pre-fusion state) and two pockets we have identified based on analysis of a recently reported late-stage fusion intermediate. Screening will employ atomic-level footprints to identify the most promising top-scoring compounds (300-400) for experimental characterization. (Aim #2) Characterize small molecule probes identified computationally by quantifying the disruption of viral entry. Top-scoring compounds (300-400) predicted to arrest entry by disrupting membrane fusion will be tested in Vero cells and human fibroblasts (HFF-1). Initial screens will be performed in a high- throughput non-replicating pseudotyped virus system (ZIKV M/E protein in an HIV particle with a luciferase reporter). Positive hits will be tested with live ZIKV infectivity assays (plaque assay and endpoint dilution assay). Experimental testing will allow prioritization of the molecules identified by virtual screening. Broader impacts of the work include increased understanding of how to target related flaviviruses including dengue, yellow fever, tick-borne encephalitis, and West Nile.